Basolateral Membrane-associated 27-kDa Heat Shock Protein and Microfilament Polymerization
1997; Elsevier BV; Volume: 272; Issue: 41 Linguagem: Inglês
10.1074/jbc.272.41.25920
ISSN1083-351X
AutoresRandolph S. Piotrowicz, Eugene G. Levin,
Tópico(s)Muscle metabolism and nutrition
ResumoThe in vivo activity of the 27-kDa heat shock protein, a barbed-end microfilament capping protein, may be localized to the plasma membrane. To investigate this putative association, bovine endothelial cells expressing the human wild type or a mutant nonphosphorylatable 27-kDa heat shock protein were subjected to subcellular fractionation and immunoblot analysis. The 25-kDa endogenous bovine homolog and both exogenous gene products partitioned with cytosolic or plasma membrane components, indicating that phosphorylation is not required for membrane association. Phorbol ester treatment resulted in phosphorylation of only membrane-associated 25-kDa and wild type 27-kDa heat shock protein and did not induce redistribution. In a second fractionation protocol, streptavidin-agarose precipitation of extracts prepared from cells biotinylated at either the apical or basal surface localized membrane 25- and 27-kDa heat shock protein exclusively to the basolateral surface. Stimulation of transfectants expressing the wild type 27-kDa heat shock protein resulted in its phosphorylation and a doubling in the amount of membrane-associated F-actin precipitated, whereas the mutant protein decreased the amount of F-actin precipitated. These data suggest that membrane-associated 25- and 27-kDa heat shock proteins inhibit the generation of basolateral microfilaments and that phosphorylation releases this inhibition. The in vivo activity of the 27-kDa heat shock protein, a barbed-end microfilament capping protein, may be localized to the plasma membrane. To investigate this putative association, bovine endothelial cells expressing the human wild type or a mutant nonphosphorylatable 27-kDa heat shock protein were subjected to subcellular fractionation and immunoblot analysis. The 25-kDa endogenous bovine homolog and both exogenous gene products partitioned with cytosolic or plasma membrane components, indicating that phosphorylation is not required for membrane association. Phorbol ester treatment resulted in phosphorylation of only membrane-associated 25-kDa and wild type 27-kDa heat shock protein and did not induce redistribution. In a second fractionation protocol, streptavidin-agarose precipitation of extracts prepared from cells biotinylated at either the apical or basal surface localized membrane 25- and 27-kDa heat shock protein exclusively to the basolateral surface. Stimulation of transfectants expressing the wild type 27-kDa heat shock protein resulted in its phosphorylation and a doubling in the amount of membrane-associated F-actin precipitated, whereas the mutant protein decreased the amount of F-actin precipitated. These data suggest that membrane-associated 25- and 27-kDa heat shock proteins inhibit the generation of basolateral microfilaments and that phosphorylation releases this inhibition. Endothelial cell F-actin exists as a dynamic and responsive microfilament cytoskeleton tightly regulated by a number of spatial and temporally regulated microfilament modulating proteins (1Gotlieb A.I. Toxicol. Pathol. 1990; 18: 603-617Crossref PubMed Scopus (50) Google Scholar, 2Cassimeris L. McNeill H. Zigmond S.H. J. Cell Biol. 1990; 110: 1067-1075Crossref PubMed Scopus (131) Google Scholar, 3Watts R.G. Howard T.H. Cell Motil. Cytoskeleton. 1992; 21: 25-37Crossref PubMed Scopus (49) Google Scholar, 4Theriot J.A. Estes J.E. Higgins P.J. Actin: Biophysics, Biochemistry and Cell Biology. Plenum Publishing Corp., New York1994: 133-145Google Scholar). One such protein, a barbed-end filament capping protein that is inhibited by its phosphorylation, is the 27-kDa heat shock protein (HSP27) 1The abbreviations used are: HSP27, human 27-kDa heat shock protein; HSP25, bovine 25-kDa heat shock protein; wtHSP27, exogenous wild type human 27-kDa heat shock protein; muHSP27 non-phosphorylatable exogenous human 27-kDa heat shock protein; PMA, phorbol 12-myristate 13-acetate; SA, streptavidin-conjugated agarose; PAGE, polyacrylamide gel electrophoresis; BAECs, bovine arterial endothelial cells; Rad, the densitometric value of an autoradiographic signal; Ag, the densitometric value of an immunostained signal from a chemiluminograph. (5Miron T. Vancompernolle K. Vandekerckhove J. Wilchek M. Geiger B. J. Cell Biol. 1991; 114: 255-261Crossref PubMed Scopus (389) Google Scholar, 6Miron T. Wilchek M. Geiger B. Eur. J. Biochem. 1988; 178: 543-553Crossref PubMed Scopus (101) Google Scholar, 7Benndorf R. Hayeß K. Ryazantsev S. Wieske M. Behlke J. Lutsch G. J. Biol. Chem. 1994; 269: 20780-20784Abstract Full Text PDF PubMed Google Scholar). The in vivo activity of HSP27 has been inferred from the effects of overexpression of the protein in cultured fibroblasts (8Landry J. Chr'tien P. Lambert H. Hickey E. Weber L.A. J. Cell Biol. 1989; 109: 7-15Crossref PubMed Scopus (582) Google Scholar, 9Lavoie J.N. Hickey E. Weber L.A. Landry J. J. Biol. Chem. 1993; 268: 24210-24214Abstract Full Text PDF PubMed Google Scholar). Expression of human HSP27 in these cells stabilized cortical F-actin microfilaments that normally disaggregate in response to heat shock, acute cytochalasin D treatment, or oxidative stress (10Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (570) Google Scholar, 11Huot J. Lambert H. Lavoie J.N. Guimond A. House F. Landry J. Eur. J. Biochem. 1995; 227: 418-427Crossref Scopus (173) Google Scholar). In unstressed cells, overexpression of HSP27 leads to increased pinocytotic activity and membrane ruffling (9Lavoie J.N. Hickey E. Weber L.A. Landry J. J. Biol. Chem. 1993; 268: 24210-24214Abstract Full Text PDF PubMed Google Scholar), processes dependent on a dynamic membrane-associated microfilament cytoskeleton (12Bar-Sagi D. Feramisco J.R. Science. 1986; 233: 1061-1068Crossref PubMed Scopus (539) Google Scholar, 13Theriot J.A. Mitchison T.J. Nature. 1991; 352: 126-131Crossref PubMed Scopus (650) Google Scholar). Whether the in vivo changes that result from the enhanced expression of HSP27 are due to the microfilament capping activity demonstrated for HSP27 in vitro has yet to be firmly established, however. HSP27 is phosphorylated by kinase activity induced by a variety of stress, cytokine and mitogenic stimuli (11Huot J. Lambert H. Lavoie J.N. Guimond A. House F. Landry J. Eur. J. Biochem. 1995; 227: 418-427Crossref Scopus (173) Google Scholar, 14Guesdon F. Freshney N. Waller R.J. Rawlinson L. Saklatvala J. J. Biol. Chem. 1993; 268: 4236-4243Abstract Full Text PDF PubMed Google Scholar, 15Ahlers A. Belka C. Gaestel M. Lamping N. Sott C. Herrmann F. Brach M.A. Mol. Pharmacol. 1994; 4: 1077-1083Google Scholar, 16Rouse J. Cohen P. Trigon S. Morange M. Alonso-Liamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1503) Google Scholar, 17Engel K. Ahlers A. Brach M.A. Herrmann F. Gaestel M. J. Cell. Biochem. 1995; 57: 321-330Crossref PubMed Scopus (54) Google Scholar). The control of HSP27 in vivo activity has been linked to its phosphorylation state, a conclusion based on results demonstrating that a non-phosphorylatable mutant (mu) HSP27, expressed in cultured cells, failed to promote the same effects promoted by the wild type (wt) HSP27 (8Landry J. Chr'tien P. Lambert H. Hickey E. Weber L.A. J. Cell Biol. 1989; 109: 7-15Crossref PubMed Scopus (582) Google Scholar, 9Lavoie J.N. Hickey E. Weber L.A. Landry J. J. Biol. Chem. 1993; 268: 24210-24214Abstract Full Text PDF PubMed Google Scholar). Evidence has been presented, however, that contradicts the reliance of HSP27 activity on its phosphorylation. For example, Knaufet al. (18Knauf U. Jakob U. Engel K. Buchner J. Gaestel M. EMBO J. 1994; 13: 54-60Crossref PubMed Scopus (124) Google Scholar) have found that overexpression of muHSP27 confers the same thermo-resistant traits as the wild type protein and that the in vitro ability of muHSP27 to act as a molecular chaperone is also not affected by the lack of phosphorylation. To investigate a putative role of HSP27 in controlling the endothelial microfilament cytoskeleton, we have generated stable transfectants of bovine arterial endothelial cells (BAECs) expressing the human HSP27 gene (wtHSP27) (19Hickey E. Brandon S.E. Potter R. Stein G. Stein J. Weber L.A. Nucleic Acids Res. 1986; 14 (4127): 4127Crossref PubMed Scopus (194) Google Scholar) or a mutagenized gene product (muHSP27) lacking the three known phosphorylation sites (9Lavoie J.N. Hickey E. Weber L.A. Landry J. J. Biol. Chem. 1993; 268: 24210-24214Abstract Full Text PDF PubMed Google Scholar). The transfected BAECs constitutively express the exogenous gene products in addition to the bovine HSP27 homolog, HSP25 (20Piotrowicz R.S. Weber L.A. Hickey E. Levin E.G. FASEB J. 1995; 9: 1079-1084Crossref PubMed Scopus (31) Google Scholar, 21Li S. Piotrowicz R.S. Levin E.G. Shyy Y.J. Chien S. Am. J. Physiol. 1996; 271: C994-C1000Crossref PubMed Google Scholar). Bovine HSP25 and human wtHSP27, but not muHSP27, are phosphorylated to similar levels by kinase activity induced in response to chemical (phorbol 12-myristate 13-acetate, PMA) or mechanical (laminar flow) stimuli (21Li S. Piotrowicz R.S. Levin E.G. Shyy Y.J. Chien S. Am. J. Physiol. 1996; 271: C994-C1000Crossref PubMed Google Scholar). The transfected clones express 20–40 ng of exogenous HSP27/20 μg of total transfectant protein, which has no effect on the expression of the endogenous HSP25 (21Li S. Piotrowicz R.S. Levin E.G. Shyy Y.J. Chien S. Am. J. Physiol. 1996; 271: C994-C1000Crossref PubMed Google Scholar). In addition, expression of either exogenous gene product does not effect the basal or stimulated level of HSP25 phosphorylation (21Li S. Piotrowicz R.S. Levin E.G. Shyy Y.J. Chien S. Am. J. Physiol. 1996; 271: C994-C1000Crossref PubMed Google Scholar). Thus, at the level of expression generated in the BAEC clones, the exogenous gene products simply add to the cellular pool of HSP25. This addition can induce phenotypic alterations. For example, expression of wtHSP27 induces accelerated growth and culture senescence of the BAECs (20Piotrowicz R.S. Weber L.A. Hickey E. Levin E.G. FASEB J. 1995; 9: 1079-1084Crossref PubMed Scopus (31) Google Scholar). Expression of exogenous HSP27 in fibroblasts results in increased cortical actin structure and an enhancement of activities dependent on the membrane-associated microfilament cytoskeleton such as pinocytosis and membrane ruffling (9Lavoie J.N. Hickey E. Weber L.A. Landry J. J. Biol. Chem. 1993; 268: 24210-24214Abstract Full Text PDF PubMed Google Scholar, 10Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (570) Google Scholar, 11Huot J. Lambert H. Lavoie J.N. Guimond A. House F. Landry J. Eur. J. Biochem. 1995; 227: 418-427Crossref Scopus (173) Google Scholar). These data suggest that a component of cellular HSP27 activity is focused to the membrane. To investigate this possibility, we performed subcellular fractionation on BAECs and transfected BAECs expressing human wtHSP27 and muHSP27. In this report, we demonstrate that a significant portion of cellular HSP25/27 fractionates with plasma membrane components, that phosphorylation is not required for this localization, and that this subpopulation of HSP25/27 is a substrate for kinase activity induced by brief treatment of the cells with phorbol ester. Using a second fractionation protocol, we further demonstrate that a portion of the membrane-associated HSP25/27 localizes to the basolateral membrane where it is substrate for HSP27 kinase activity. Concurrent with phorbol ester-induced HSP25/27 phosphorylation is the generation of additional membrane-associated F-actin. Expression of muHSP27 (which cannot be phosphorylated) inhibits the generation of additional membrane-associated F-actin. These data are consistent with the demonstrated in vitro functioning of HSP27 (i.e.that non-phosphorylated HSP27 inhibits actin polymerization and the phosphorylation abrogates this activity) (7Benndorf R. Hayeß K. Ryazantsev S. Wieske M. Behlke J. Lutsch G. J. Biol. Chem. 1994; 269: 20780-20784Abstract Full Text PDF PubMed Google Scholar). These data suggest that membrane-associated HSP25/27 may play a role in the regulation basolateral membrane-associated microfilament dynamics. Low passage bovine pulmonary arterial endothelial cells were cultured under 5% CO2 in Dulbecco's modified Eagle's media (BioWhittaker, Inc.) containing 25 mm HEPES and supplemented with 10% fetal calf serum (Intergen) and 1 mm each of sodium pyruvate, penicillin, streptomycin, and nonessential amino acids. Cells were plated at a density of 0.7–2 × 104cells/cm2 and passaged when confluent (approximately 1 × 105/cm2). All culture reagents, except where otherwise noted were obtained from BioWhittaker, Inc. Stable clonal cells lines of BAECs were prepared with modifications of what has been previously described (20Piotrowicz R.S. Weber L.A. Hickey E. Levin E.G. FASEB J. 1995; 9: 1079-1084Crossref PubMed Scopus (31) Google Scholar). Briefly, low passage BAECs, isolated from pulmonary arteries, were transfected with a plasmid containing a genomic clone of human HSP27 (8Landry J. Chr'tien P. Lambert H. Hickey E. Weber L.A. J. Cell Biol. 1989; 109: 7-15Crossref PubMed Scopus (582) Google Scholar), a plasmid containing that clone subjected to site-directed mutagenesis in which the codons for the principal sites of HSP27 phosphorylation (Ser15, Ser78, and Ser82) were altered to encode glycine residues (9Lavoie J.N. Hickey E. Weber L.A. Landry J. J. Biol. Chem. 1993; 268: 24210-24214Abstract Full Text PDF PubMed Google Scholar) or vector plasmid without insert (pBluescript, pBS) using the cationic lipid LipofectAMINE (Life Technologies, Inc.). The cells were plated at a density of 4 × 104/cm2 in 96- and 6-well plates. A plasmid conferring neomycin resistance, pCDM8neo, was co-transfected at a one-tenth molar ratio for the purpose of antibiotic selection. Transfected populations were grown for 72 h at 37 °C under 5% CO2 in Dulbecco's modified Eagle's media containing 25 mm HEPES and 4.0 g/liter glucose supplemented with 1 mm sodium pyruvate, 1 mm nonessential amino acids, 1 mmpenicillin/streptomycin, and 10% fetal calf sera (Intergen Corp.). The cells grown in the 6-well plates were screened for transfection efficiency via immunoblot analysis and immunofluorescence microscopy using the anti-HSP27 monoclonal antibody G3.1 (StressGen, Inc.). Selection media, culture media containing 700 μg/ml geneticin (Life Technologies, Inc.), was then added to the 96-well plates. Media were changed every 3 days as non-transfected cells died. After 2 weeks of culture in selection media, the 96-well plates were viewed via phase contrast microscopy, and the wells containing single colonies were noted. These clones were subcultured in selection media. Following this protocol, a single transfection performed in three 96-well plates generated 15–25 clones. Clones developed from several different transfections were used for this study and demonstrated by densitometric analysis of immunoblots on which recombinant human HSP27 (StressGen, Inc.) was analyzed to contain approximately 20–40 ng of exogenous HSP27/20 μg of total cellular protein. Cells were cultured in complete media prepared with phosphate-free Dulbecco's modified Eagle's media (Sigma) containing 100 μCi/ml [32P]orthophosphate (NEN Life Science Products; 900 Ci/mmol) for 45 min at 37 °C. At this point, 100 nmphorbol 12-myristate 13-acetate (PMA, Sigma) or vehicle was added directly to the cultures and incubated for an additional 10 min. The cells were then rinsed three times with Dulbecco's phosphate-buffered saline (BioWhittaker) and used for subcellular fractionation or surface biotinylation. Transfected BAECs were fractionated using a slightly modified protocol from that described (22Gazitt Y. Friend C. Cancer Res. 1981; 41: 1064-1969PubMed Google Scholar). Briefly, 6–10 × 106 BAECs or transfected BAECs were radiophosphorylated as described, stimulated with 100 nmPMA for 10 min, and then released from the substratum by trypsinization. After quenching the trypsinization with media containing 10% fetal calf sera, the cells were centrifuged at 200 × g and washed three times in Dulbecco's phosphate-buffered saline. The final cell pellet was resuspended in a cold hypotonic buffer consisting of 2 mm Tris, pH 8.0, 0.2 mm adenosine triphosphate, 2 mmCaCl2, 10 mm sodium fluoride, 1 mmsodium vanadate, 100 μg/ml leupeptin, 1 mm benzamadine hydrochloride, and 1 mm phenylmethylsulfonyl fluoride. For efficient release and subsequent fractionation, hypotonic buffer was added at a ratio of at least 1 ml to approximately 2–3 × 106 cells. After incubation on ice for 15 min, the resultant nucleated ghosts were pelleted by centrifugation at 500 × g for 5 min at 4 °C. The hypotonic lysate was collected, and a volume of 5 m NaCl was added to yield a final concentration of 145 mm. The lysate was then cleared by ultracentrifugation at 100,000 × g for 1 h at 4 °C. The pelleted nucleated ghosts were then resuspended in the hypotonic buffer, pelleted, and washed twice more. The final supernatant was removed, and a volume of 0.1% Nonidet P-40 (v/v) in the hypotonic buffer was added to the pelleted nucleated ghosts. The nucleated ghosts were then subjected to homogenization using a Dounce homogenizer. The resultant homogenate was cleared of nuclei by centrifugation at 500 × g for 5 min and then cleared of endosomal material and mitochondria by ultracentrifugation at 100,000 × g for 1 h at 4 °C. A volume of 5m NaCl was added to the resultant homogenate to yield a final concentration of 145 mm. Protein content of both the hypotonic releasate and Nonidet P-40 homogenate was determined using the bicinchoninic acid method (Pierce). Apical and basolateral biotinylation of cells was performed essentially as described (23Ellis J.A. Jackman M.R. Luzio J.P. Biochem. J. 1992; 283: 553-560Crossref PubMed Scopus (21) Google Scholar). For apical biotinylation, transfected clones were cultured in tissue culture wells. For basal biotinylations, transfectants were cultured in 6.8-cm2tissue culture-treated Transwell culture inserts with 3-micron pores. Cells were cultured 1–2 weeks past the point of confluence. For each experiment, fresh succinimidyl-6-(biotinamido)hexanoate (Pierce) was dissolved in chilled 50 mm NaHCO3, pH 8.2, 145 mm NaCl to yield a concentration of 55 μg/ml. The cells were washed 3 times in prechilled biotinylation buffer; the biotin solution was added and incubated with the cells at 4 °C for 30 min. For the basal biotinylation, the biotin solution was stirred in the lower chambers of the Transwell plate with microstir bars. After biotinylation, the cell monolayers were washed 3 times with Dulbecco's phosphate-buffered saline and lysed in 0.5% Triton X-100 in 10 mm imidazole, pH 7.15, 40 mm KCl, 10 mm EGTA containing 10 mm benzamidine, 0.5 mm phenylmethylsulfonyl fluoride, 100 μg/ml leupeptin, 2 mm sodium vanadate, and 0.5 mm sodium fluoride, a buffer that stabilizes and preserves F-actin (3Watts R.G. Howard T.H. Cell Motil. Cytoskeleton. 1992; 21: 25-37Crossref PubMed Scopus (49) Google Scholar). Cells were lysed in a volume of 200 μl per Transwell insert. Lysis and all subsequent steps were performed at room temperature. The lysates were briefly centrifuged at 500 × g for 2 min, and a sample was saved for protein determination and immunoblot analysis. The remaining portion of the lysates was incubated with streptavidin-conjugated agarose (SA, Pierce) equilibrated to the lysis buffer at a ratio of 50 μl of prepared SA-agarose beads per 200 μl of lysate. After incubating for 30 min at room temperature while gently mixing, unbound material was removed by three consecutive washes in 10 bead volumes of lysis buffer. Cellular material was eluted off the SA-agarose by boiling in Laemmli SDS-PAGE sample buffer for 3 min. Samples were reduced by the addition of 5% β-mercaptoethanol and then analyzed for the presence of HSP27 and actin by immunoblot analysis. In some experiments, transfected BAECs were radiophosphorylated (as described above) prior to basal biotinylation. In every experiment, serially diluted Triton X-100 lysates were also subjected to immunoblot and autoradiographic analyses for the purpose of demonstrating linearity of the immunoreactive and autoradiographic signals (see below). Cell subfractions, lysates, or streptavidin eluates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions on 12% (w/v) polyacrylamide gels (24Laemmli U.K. Nature. 1970; 277: 680-685Crossref Scopus (207233) Google Scholar). Upon completion of electrophoresis, proteins were transferred to nitrocellulose membranes that were then blocked for 1 h at room temperature in 5% nonfat milk in 20 mmTris, pH 7.4, 145 mm NaCl with 0.05% Tween 20 (TTBS). The membranes were then incubated with dilutions of rabbit antisera raised against murine HSP25 (StressGen), a murine monoclonal antibody (G3.1, StressGen) specific for human HSP27 or a monoclonal antibody specific for non-muscle β-actin (clone AC-15, Sigma) in TTBS containing 1.0 mg/ml bovine serum albumin. Bound antibody was detected using donkey anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase (Jackson Laboratories) and the enhanced chemiluminescence reagent (Amersham Corp.). The stained immunoblots were placed against x-ray film (BioMax, Eastman Kodak Co.) to generate chemiluminographs. Densitometry of chemiluminographs and autoradiographs of immunoblots was performed with a Stratagene Eagle Eye II computerized digital camera and Eagle Sight 2.0 software. Densitometric data are generated as integrated density units. Chemiluminographic signals for immunoreactive material generated in the experiments presented in this report were within a linear range determined by analyzing serially diluted Triton lysates of BAECs or wtHSP27-expressing BAECs (Fig. 1, panels A and B) with the primary antibodies recognizing HSP25 (rabbit antisera raised to murine HSP25, StressGen, Inc.), HSP27 (the monoclonal antibody G3.1, StressGen, Inc.), and β-actin (clone AC-15, Sigma). Likewise, autoradiographic signal intensities were within the linear range of exposures as defined by densitometric analysis of the autoradiograph of an immunoblot on which serial dilutions of a Triton X-100 lysate prepared from radiolabeled wtHSP27-BAECs were analyzed (Fig. 1,panels A and B). For fractionation experiments of radiophosphorylated BAECs, the autoradiographic integrated densities (Rad) of cytosolic HSP25/27 and membrane-associated HSP25/27 were determined for quiescent and PMA-stimulated BAECs. These values were divided by the integrated densitometric values of the immunostained HSP25/27 bands on chemiluminographs (i.e. antigen intensity, Ag). The resultant Rad/Ag ratios were averaged and presented with the calculated standard deviations. Overexpression of HSP27 in transfected cell lines generates enhanced cortical F-actin structure and alters cellular processes dependent on a dynamic membrane-associated microfilament cytoskeleton (e.g.pinocytosis and membrane ruffling) (9Lavoie J.N. Hickey E. Weber L.A. Landry J. J. Biol. Chem. 1993; 268: 24210-24214Abstract Full Text PDF PubMed Google Scholar, 10Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (570) Google Scholar, 11Huot J. Lambert H. Lavoie J.N. Guimond A. House F. Landry J. Eur. J. Biochem. 1995; 227: 418-427Crossref Scopus (173) Google Scholar), suggesting a functional association with the plasma membrane. To determine if endothelial cell HSP25 localizes to this cell compartment, BAECs were fractionated by sequential hypotonic lysis, homogenization in the presence of Nonidet P-40, and differential centrifugation (22Gazitt Y. Friend C. Cancer Res. 1981; 41: 1064-1969PubMed Google Scholar). Using this protocol, BAE cell fractions enriched for nuclear (N), plasma membrane (P), microsomal (M), and cytosolic components (C) were generated and subjected to immunoblot analysis using rabbit antisera raised against murine HSP25 (Fig. 2, panel a). HSP25 primarily fractionated with either the Nonidet P-40 homogenate containing solubilized plasma membrane components (P) or the hypotonic lysate containing cytosolic proteins (C). The protocol employed efficiently fractionates the plasma membrane compartment from the cytosolic compartment as demonstrated by the lack of anti-glucose-6-phosphate dehydrogenase (G6PDH) immunoreactive material in the plasma membrane fraction (P) but staining of immunoreactive material in the cytosolic (C) fraction (Fig. 2, panel b). Conversely, cytosolic material was not contaminated with plasma membrane components as indicated by the lack of anti-integrin β3 immunoreactivity in the cytosolic fraction but positive staining of a band with the appropriate apparent molecular mass (100 kDa) in the plasma membrane fraction (Fig.1, panel C, lane P). To determine whether phosphorylation of HSP27 is necessary for the partitioning of this protein into the membrane fraction, clones of BAECs expressing HSP27 or the non-phosphorylatable muHSP27 were also fractionated by this method and analyzed with the monoclonal antibody G3.1 that is specific for the human protein. Both wtHSP27 (Fig.2, panel d, lanes 1–3) and muHSP27 (Fig. 2, panel d, lanes 4 and 5) partitioned with the plasma membrane fraction indicating that phosphorylation of HSP27 is not required for the association of HSP25/27 with the membrane components. In panel d, a greater percentage of the plasma membrane fractions was subjected to analysis than the hypotonic lysates containing cytosolic components. This accounts for the greater signals obtained for the HSP25/27 in the plasma membrane fractions of the individual clones. Cell fractionation experiments detailed above were performed, and the relative antigen level in each fraction was determined by immunoblot analysis. The antigenic signals obtained for the membrane-associated or cytosolic HSP25/27 from the fractionated transfectants fell within the linear range of immunodetection (Fig. 1), allowing the comparison of signal intensities. The relative amounts of HSP25/27 in the cytosolic fractions and the plasma membrane fractions of BAECs, wtHSP27-, or muHSP27-expressing BAECs were calculated from the densitometric signals obtained from the immunoblot and the percentage of each fraction analyzed. The average percentages of total cellular HSP25, wtHSP27, and muHSP27 that partitioned with the membrane fractions were determined to be 27 ± 9 (n = 14), 26 ± 12, and 24 ± 15%, respectively. Thus a significant portion of the endogenous HSP25 and the exogenous HSP27 gene products fractionate with membrane components. These results are not due to the nonspecific trapping of large oligomeric complexes containing HSP25/27 since glucose-6-phosphate dehydrogenase, which also exists in hetero-oligomeric complexes exhibiting molecular mass of up to 250 kDa (25Hogeboom G.H. Schneider W.C. J. Biol. Chem. 1950; 186: 417-427Abstract Full Text PDF PubMed Google Scholar, 26Glock G.E. McLean P. Biochem. J. 1953; 55: 400-408Crossref PubMed Scopus (725) Google Scholar), was not detected in the plasma membrane fractions. The microfilament capping activity of HSP25, which inhibits actin polymerization in vitro, is abrogated by HSP25 phosphorylation (7Benndorf R. Hayeß K. Ryazantsev S. Wieske M. Behlke J. Lutsch G. J. Biol. Chem. 1994; 269: 20780-20784Abstract Full Text PDF PubMed Google Scholar). To investigate whether membrane HSP25 is phosphorylated in response to kinase activity induced by phorbol esters, BAECs were radiolabeled with 32Pi, stimulated with PMA, and then fractionated by the above procedure. To evaluate the relative levels of HSP25 phosphorylation in the two cellular compartments, autoradiographic and immunoblot analyses of the cytosolic and plasma membrane fractions obtained from PMA-stimulated,32Pi-labeled BAECs were performed (Fig.3, panel A). Because two-dimensional isoelectric focusing/SDS-PAGE demonstrated that no other phosphoproteins in endothelial cell lysates co-migrate in the second dimension with phospho-HSP25 (27Levin E.G. Santell L. J. Immunol. 1991; 146: 3772-3778PubMed Google Scholar, 28Levin E.G. Santell L. J. Biol. Chem. 1991; 266: 174-181Abstract Full Text PDF PubMed Google Scholar), one-dimensional SDS-PAGE was deemed adequate for this study. Immunostaining for HSP25 demonstrated equivalent loading of HSP25 in the membrane and cytosolic fractions for both stimulated (+) and non-stimulated (−) cells. PMA stimulation did not result in a change in the relative distribution of cellular HSP25 between the two compartments (Fig. 3, panel A). The percentage of cellular HSP25 that partitioned with the plasma membrane fraction was determined to be 37 ± 9 and 35 ± 10% (n = 5) for non-stimulated and stimulated BAECs, respectively. The autoradiographic intensity of the cytosolic HSP25 did not increase as a result of PMA stimulation (Fig. 3,panel A). In contrast, the autoradiographic intensity of the membrane-associated HSP25 exhibited a 2.7-fold increase upon treatment with PMA. To compare the relative levels of PMA-induced phosphorylation of membrane-associated HSP25 to cytosolic HSP25, the autoradiographic integrated densities (Rad) of cytosolic HSP25 and membrane-associated HSP25 were determined for quiescent and PMA-stimulated BAECs and then normalized to the chemiluminograph antigenic integrated densitometric values (Ag) obtained for the HSP25 immunostained bands. The resultant Rad/Ag ratios were averaged and presented with the calculated standard deviations in Fig. 3, panel B. The basal phosphorylation level of the plasma membrane-associated HSP25 was slightly higher than that of the cytosolic HSP25. Upon P
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